This proposal, entitled Vascularization of Polymeric Tissue Beds, seeks to address the broad Challenge Area of (11) Regenerative Medicine, and the specific Challenge Topic of vascular networks in engineered tissues: 11-EB-101. The major impediment to progress in the field of tissue engineering and regenerative medicine is the lack of effective vascularization in tissue-engineered constructs capable of timely delivery of oxygen and nutrients, and removal of waste products. To address this major challenge, the proposed research seeks to elucidate mechanisms that direct vasculogenesis and angiogenesis by using a cellular platform that can probe the material parameters, and a modular synthetic platform that can be tuned to enhance vasculogenesis. The overall goal is to create a prevascularlized tissue bed by tuning a cellular platform to enhance homing and remodeling, and a polymeric platform that is conducive for vasculogenesis. The central hypothesis is that a tissue engineered construct conducive to vascularization requires the capacity 1) to provide endothelial cell specific attachment, 2) for local tissue remodeling, and 3) to recruit endothelial cells through cytokine signaling. To investigate this hypothesis, we propose four specific Aims: (1) to engineer cellular attachment and matrix remodeling functions into embryonic stem cell derived endothelial cells. We plan to overexpress alpha-V and beta-3, ([unreadable]v[unreadable]3) or alpha-5 and beta-1 ([unreadable]5[unreadable]1) integrins, to enhance the cell's capacity for attachment in an ECM-mimetic polymer network, and overexpress MMP1 to enhance the cell's capacity for tissue remodeling. (2) To engineer a PEG-based ECM-mimetic hydrogel, which incorporates integrin binding peptides (-RGD- or -CRRETAWAC- which has high affinity and selectivity for [unreadable]v[unreadable]3 and [unreadable]5[unreadable]1) for cell attachment;a collagenase-sensitive peptide -GPQGIAGQ- for EC mediated biodegradation, and entrapped nanoparticles that provide a reservoir for controlled release of angiogenic factors (VEGF). (3) To integrate and tune the cellular and polymeric platforms, to achieve optimal vasculogenic and angiogenic responses, as determined by vessel counts and 3D vessel reconstruction. Clones expressing varying levels of [unreadable]v[unreadable]3 and [unreadable]5[unreadable]1 integrins will be tested with ECM-mimetic hydrogels with a range of peptide ligand density. Clones expressing varying levels of MMP1 will be tested with hydrogels varying in PEG molecular weight. The effect of VEGF secretion will be evaluated by quantitative measures of EC progenitor migration. (4) To evaluate prevascularization of the cellular and polymeric platforms in vivo, optimized scaffolds will be implanted into a mouse subcutaneous model of vascularization and compared with Matrigel positive controls, and monitored by in vivo imaging techniques (bioluminescence and PET). Successful completion of this research will generate fundamental insights into the requirements needed for developing vascular networks in engineered tissue constructs and lay a basic foundation for facilitating clinical translation into engineered tissues. PUBLIC HEALTH RELEVANCE: Tissue engineering is an approach that seeks to augment or replace the function of failing organs, and offers the potential for major beneficial impact on human health. However, the major impediment to progress in this field is the lack of effective vascularization of the tissue-engineered constructs capable of timely delivery of oxygen and nutrients and removal of waste products. To address this major challenge, the proposed research seeks to evaluate the mechanisms that direct angiogenesis in parallel with using a cellular platform that can probe the material parameters, and a modular synthetic platform that can be tuned to improve vasculogenesis. Successful completion of the research will generate fundamental insights into the requirements needed for developing vascular networks in engineered tissue constructs and facilitate translation into clinical applications.